Genome-wide target analysis of Shh-activated transcription network in limb bud

我们的长期目标是解开基因调控和表达的早期模式与结构的最终实现之间的联系，以作为信号网络如何协调复杂组织形成的范例。为了实现这一目标，我们正在开发几个相结合的遗传学和基因组学，蛋白质组学的方法来研究转录因子和调节级联在肢体发育过程中运作，最终目的是阐明调控层次之间的早期诱导前后（AP）模式和不同的数字（不同数量，长度和形状的趾骨）的形态发生。了解转录因子如何在正常发育过程中协调生长和形态发生，将促进我们对如何治疗遗传疾病和癌症的理解，这些疾病和癌症是在这些调控成分突变或异常表达时出现的。Hoxd基因如何指导手指的特征（如关节的数量，形状，大小）以及Hoxd和Gli 3靶标之间的关系？了解这种三维结构如何形成通常与了解器官发生是如何实现的有关。最近已经报道了一些5 Hoxd体内靶标（Shh增强子、Hand 2启动子），尽管没有很好地表征。定义5 Hoxd功能的时间窗对于选择ChIP分析的肢体分期非常重要。我们的遗传研究结果表明，在不同的时间删除Hoxd基因功能的影响表明，这些基因调节手指形态和关节形成在相对较晚的阶段，手指的发展，当前体射线已经开始出现。我们已经开发了几种针对ChIP的抗体，并且我们与Steve Vokes（U.德克萨斯州，奥斯汀）。这种可诱导的转基因也将促进靶点的生物学验证，沿着我们已经在实验室中拥有的条件性敲除等位基因。结果将与Vokes实验室的预期结果相关联，Vokes实验室也在分析Gli 3靶点，并且已经为我们提供了一种表位标记的Gli 3表达小鼠系。确定Hoxd和Gli 3的目标将提供深入了解共调节基因和Gli 3-Hoxd的作用，以及阐明Hoxd基因在肢体形态发生中的晚期效应。还将分析肢体中由Hoxd和Gli 3调节的转录网络与形成两种不同类别的Shh通路靶点的关系，这些靶点需要瞬时或持续的信号传导才能稳定激活。通过这种方式，我们希望揭示导致具有不同数量的节段和关节的定义的手指形态形成的调节级联。Gli和Hox基因在某些癌症中也异常共表达，可能有助于其发病机制，这些研究也将揭示它们在这些背景下可能的作用。

Our long term goal is to unravel the steps linking early patterns of gene regulation and expression with the ultimate realization of structure to serve as a paradigm for how signaling networks orchestrate the formation of a complex tissue. To accomplish this, we are developing several combined genetic and genomic, and proteomic approaches to study transcription factors and regulatory cascades operating during limb development with the ultimate aim of elucidating the regulatory hierarchy between early induction of antero-posterior (AP) pattern and the morphogenesis of distinct digits (with different numbers, lengths and shapes of phalanges). Learning how transcription factors orchestrate growth and morphogenesis during normal development will advance our understanding of how to treat genetic diseases and cancers that arise when such regulatory components are either mutated or expressed abnormally. How do Hoxd genes instruct features of digit identity (such as numbers of joints, shape, size) and what is the relation between Hoxd and Gli3 targets? Learning how this 3-dimensional structure forms will be generally relevant for understanding how organogenesis is achieved. A few 5Hoxd in vivo targets have been reported recently (Shh enhancer, Hand2 promoter), albeit not well-characterized. Defining time windows for 5Hoxd functions will be important in choice of limb stages for ChIP analysis. Our genetic results examining effects of removing Hoxd gene function at different times indicates that these genes regulate digit morphology and joint formation at relatively late stages of digit development, when precursor rays for digits have begun to appear. We have developed several antibodies for ChIP, and we have successfully engineered an epitope-tagged Hoxd13 conditional transgene allele for ChIP with anti-tag in collaboration with Steve Vokes (U. Texas, Austin). This inducible transgene will also facilitate biological validation of targets, along with a conditonal knock-out allele that we already have in the lab. Results will be correlated together with anticipated results from Dr. Vokes lab, who is also analyzing Gli3 targets, and who has made an epitope-tagged Gli3 expressing mouse line available to us. Identifying Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of Hoxd genes in limb morphogenesis. The transcriptional network regulated by Hoxd and Gli3 in the limb will also be analyzed in relation to Shh-pathway targets that form two distinct classes, requiring either transient or sustained signaling for their stable activation. In this manner, we hope to uncover the regulatory cascade leading to formation of defined digit morphologies with distinct numbers of segments and joints. Gli and Hox genes are also aberrantly co-expressed in some cancers and may contribute to their pathogenesis, and these studies will also shed light on their possible roles in these contexts.